Marcelo A. Dankert

Xanthan Induces Plant Susceptibility by Suppressing Callose Deposition

Xanthan is the major exopolysaccharide secreted by Xanthomonas spp. Despite its diverse roles in bacterial pathogenesis of plants, little is known about the real implication of this molecule in Xanthomonas pathogenesis. In this study we show that in contrast to Xanthomonas campestris pv campestris strain 8004 (wild type), the xanthan minus mutant (strain 8397) and the mutant strain 8396, which is producing truncated xanthan, fail to cause disease in both Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) plants. In contrast to wild type, 8397 and 8396 strains induce callose deposition in N. benthamiana and Arabidopsis plants. Interestingly, treatment with xanthan but not truncated xanthan, suppresses the accumulation of callose and enhances the susceptibility of both N. benthamiana and Arabidopsis plants to 8397 and 8396 mutant strains. Finally, in concordance, we also show that treatment with an inhibitor of callose deposition previous to infection induces susceptibility to 8397 and 8396 strains. Thus, xanthan suppression effect on callose deposition seems to be important for Xanthomonas infectivity.

Evidence for a role for the gumB and gumC gene products in the formation of xanthan from its pentasaccharide repeating unit by Xanthomonas campestris

The biosynthesis of the extracellular polysaccharide xanthan in Xanthomonas campestris pv. campestris is directed by a cluster of 12 genes, gumB-gumM. Several xanthan-deficient mutants of the wild-type strain 8004 have previously been described which carry Tn5 insertions in this region of the chromosome. Here it is shown that the transposon insertion in one of these mutants, strain 8397, is located 15 bp upstream of the translational start site of the gumB gene. EDTA-treated cells of strain 8397 were able to synthesize the lipid-linked pentasaccharide repeating unit of xanthan from the three nucleotide sugar donors (UDP-glucose, GDP-mannose and UDP-glucuronic acid) but were unable to polymerize the pentasaccharide into mature xanthan. A subclone of the gum gene cluster carrying gumB and gumC restored xanthan production to strain 8397 to levels approximately 28% of the wild-type. In contrast, subclones carrying gumB or gumC alone were not effective. These results are discussed with reference to previous speculations, based on computer analysis, that gumB and gumC are both involved in the translocation of xanthan across the bacterial membranes.

Lipid-linked intermediates in the biosynthesis of xanthan gum

Pyruvic acid acetal and O-acetyl groups are also present in differing proportions [ 1,2]. Although thousands of tons of xanthan gum are produced in a year, because of its industrial applications [2], nothing has been reported about its biosynthesis. Evidence on the mechanism of the in vitro assembly of this polysaccharide is described in this communication: UDP-Glc, GDP-Man and UDP-GlcUA sequentially donate their sugar moieties to a lipidic acceptor to form a pentasaccharide-P-P-lipid which is subsequently polymerized into xanthan gum. The role of polyprenyl phosphosugars was first described in bacterial lipopolysaccharide [3] and murein [4] biosynthesis, and later on extended to capsular polysaccharides [S ,6], teichoic acids [7,8] and other polysaccharides closely associated to the bacterial cell body [9,10]. This is the first report on the participation of lipidlinked sugars in the biosynthesis of an exopolysaccha. ride freely liberated into the culture medium.

A xanthan-gum-like polysaccharide from Acetobacter xylinum

SUMMARY: A new exopolysaccharide, secreted in addition to cellulose, has been isolated from the culture medium of Acetobacter xylinum NRRL B42. This polysaccharide, for which the name acetan is proposed, contains glucose, mannose, glucuronic acid and rhamnose in a molar ratio of 4:1:1:1. On the basis of methylation, thin-layer, paper and gas-liquid chromatography, paper electrophoresis and mass spectrometry studies of the degradation products obtained by total and partial hydrolysis and acetolysis of acetan, the following structure is proposed for its repeating unit: Since our previous work with this strain demonstrated the in vitro synthesis of a lipid-linked heptasaccharide with the same structure, the possibility of acetan being the result of its polymerization is discussed. One to two O-acetyl residues per repeating unit are also present in positions not yet determined.

SEPARATION OF SUGAR NUCLEOTIDES, PHOSPHORIC ESTERS AND FREE SUGARS BY PAPER CHROMATOGRAPHY WITH SOLVENTS CONTAINING BORATES OF ORGANIC BASES.

Abstract Four different chromatographic solvents containing borates of organic bases are described. Two of them permit the separation of sugar nucleotides differing only in the sugar moiety and give also a good resolution for some sugar 1-phosphates. The other two systems permit, in a single run, the separation of a group of sugars which is usually difficult to achieve.

Structural studies of acetan, an exopolysaccharide elaborated by Acetobacter xylinum

The exopolysaccharide acetan, elaborated by Acetobacter xylinum, has been investigated. The polysaccharide and a heptasaccharide, obtained on enzymic hydrolysis, corresponding to the repeating unit were characterised by sugar and methylation analysis and by NMR spectroscopy and MS. It is concluded that the polysaccharide is composed of repeating units with the following structure. [formula: see text] The polysaccharide further contains approximately two O-acetyl groups per repeating unit, which have not been assigned, but it appears that they are on primary locations.

A glucose acceptor in plants with the properties of an α-saturated polyprenyl-monophophate

Abstract Organic solvent extracts from plants were fractionated by DEAE cellulose column chromatography. Fractions were obtained which acted as glucose acceptors using UDP-Glc as glucose donor and enzymes from both plant and animal origin. The acceptors and their glucosylated derivatives were compared with dolichyl-monophosphate and ficaprenyl-monophosphate by chromatographic and degradative techniques. These tests showed that the acceptors had the properties of α-saturated polyprenyl-monophosphates.

Xanthan cum biosynthesis pyruvic acid acetal residues are transferred from phosphoenolpyruvate to the pentasaccharide-P-P-lipid

Summary Xanthan gum is a complex polysaccharide produced by Xanthomonas campestris , composed of pentasaccharide repeating units containing mannose, glucose and glucuronic acid (in the ratio 2:2:1) and pyruvic acid acetal and 0-acetyl residues in differing proportions Jansson et al. (1975) Carbohyd.Res. 45, 275–282). Previous work (Ielpi et al. FEBS Lett., in press) has shown that the pentasaccharide is first assembled on a pyrophosphate lipid and then polymerized to xanthan gum. It is now shown, using EDTA-treated cells and different combinations of the adequate [ 14 C]labeled donors, that phosphoenol pyruvate provides the acetal residues and that the transfer occurs on the terminal mannose of the pentasaccharide-P-P-lipid, as judged by the solubility properties and DEAE-cellulose column chromatography of the compounds formed as well as by the behaviour of the substances liberated by mild acid and alkaline treatments and Smith degradation. The subsequent polymerization process leads to pyruvylated xanthan gum.

Adenosine diphosphate glucose: Orthophosphate adenylyltransferase in wheat germ

Abstract An enzyme has been isolated from wheat germ which catalyzes the reaction: ADP-sugar + inorganic phosphate → ADP + sugar phosphate. Maximal activity was found at pH 8–9. The equilibrium of the reaction seems to be completely displaced towards ADP formation. The enzyme acts on ADP-glucose and deADP-glucose and more slowly, on ADP-xylose and ADP-β-glucose. Evidence is presented indicating that inorganic phosphate is incorporated in the terminal position of the nucleotide. Arsenate can be substituted for inorganic phosphate, AMP being the final product.

Lipid-linked Intermediates and the Synthesis of Acetan in Acetobacter xylinum

Summary: Several strains of Acetobacter xylinum were screened for in vivo cellulose and acetan production, and for in vitro synthesis of a prenyl-diphosphate-hexasaccharide, using UDP-Glc, UDP-GlcA and GDP-Man as sugar donors. The lipid-bound saccharide was synthesized only by acetan-producing strains. Previous work has shown that the in vitro-synthesized lipid-linked saccharides have the same structure as the acetan repeating unit. The present results strongly suggest a precursor-product relationship. The strains that produced acetan lost their ability to do so by ageing of the culture.